The SIR provided L-Aspartic Acid (4- 13C) , HOO*CCH2CH(NH2)COOH; 500mg L-Tyrosine (phenol-3,5- 13C) , HOC6H4CH2CH(NH2)COOH; 500mg L-[RING-"C6]Tyrosine; 500mg Hemoglobin is an a2/b2 tetramer that cooperatively binds oxygen, undergoing a structural rearrangement in the process. Its allosteric transition between two states has been widely accepted, but many aspects of hemoglobin behavior still remain a mystery. Among the unknowns is the specific mechanism of the Bohr effect which promotes the release of oxygen under acidic conditions. This proposal describes the application of istopic labeling, NMR, and IR to investigate the biochemical significance and character of a specific hydrogen bond which has been suggested to have a significant role in the R to T transition in hemoglobin and may contribute to the Bohr effect. This H-bond between Tyra42 and Asp b99 at the alb2 interface is thought to be critical in stabilizing the T state based on data from several experimental fields. Based on UV resonance Raman and FIIR of unlabeled protein, it appears that Asp b99 may be the H-bond donor of the pair and thus, may be involved in the Bohr effect. Experiments on strategically labeled hemoglobin should allow the exact composition of the H-bond to be determined. Once this is determined, pH dependent studies could be conducted to investigate the behavior of specific residues in response to changes in acidity. Other